Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay

Abstract

We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ∼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.

Keywords: Isotopic labeling; Liposomes; Membrane proteins; Reconstitution; Solid-state NMR.

Figure 1

SDS-PAGE analysis of KcsA during purification is shown. Panel A shows results from Nickle column on the lysate from pask90 expression and panel B shows results using the PQE-60 expression system. The band around 50 KDa is the KcsA tetramer. Weak bands at ~100 KDa and ~20 KDa correspond to octamers and monomers respectively. Panel A lanes: 1-marker 2-cell lysate supernatent, 3,4 – consecutive 50 mM imidazole washes, 5,6 - 100 mM imidazole elution 7– 150 mM imidazole elution, 8- 200 mM imidazole elution, 9- 250 mM imidazole elution, 10- pure KcsA standard. Panel B lanes: 1,3,4- 50 mM imidazole washes, 2-cell lysate supernatant, 5–6 – 100 mM imidazole elution, 7 – 150 mM imidazole elution, 8– 200 mM imidazole elution, 9–10 – 250mM imidazole elution.

Figure 2

Cryo-EM images of KcsA proteoliposomes in DOPE/DOPS lipid membranes are shown. In (A) an image of the sample before magic angle spinning shows predominantly small, unilamellar vesicles. Panel (B) shows an aliquot of the sample after magic angle spinning at 14 KHz for several days. The spinning causes these the vesicles to be converted into large multilamellar vesicles, but the bilayer remains intact.

Figure 3

Solid-state NMR spectra of ~ 10 mg of uniformly ¹³C-¹⁵N labeled KcsA at 50 mM K⁺, pH 7.5 measured on a 750 MHz instrument are shown. Panel A shows a C1D CP spectrum acquired with 32 scans acquired at an MAS of 12 KHz and a sample temperature of 0–10 C. Panel B shows a N1D CP spectrum acquired with 128 scans at an MAS of 14 KHz and the same temperature. Panel C shows the Cα-Cβ region of a ¹³C-¹³C correlation spectrum of KcsA using the DARR mixing sequence and 15ms mixing. The spectra show well-ordered narrow lineshapes for several resonances

Figure 4

Electrophysiology results on Uniformly ¹³C-¹⁵N labeled KcsA (U-KcsA). Channels were reconstituted into 3POPE: 1POPG liposomes as described in the paper. Currents were recorded in symmetrical 100 mM K⁺. In panel A, a single channel trace of U-KcsA at 100 mV is shown. Traces were low pass filtered at 500 Hz. In Panel B the Current-voltage curve for U-KcsA (black circles. −175 mV to 175 mV) and WT KcsA (open circles, −175 mV to 375 mV) is shown. The current-voltage curve is also measured in the presence of 10 mM intracellular Na⁺ (red circles, −175 mV to 375 mV) to confirm selectivity. The data show that our protocols yield selective and functional K⁺ channels.